Vaccine protection against smallpox is based on shared antigenic specificities between vaccinia virus (VACV), the vaccine and variola virus (VARV) the causative agent of smallpox. Understanding how the level of cross-reactivity between poxviruses affects protection and knowing which factors shape the specificity of immune responses to poxviruses are important for the design of new smallpox vaccines. Recently, several CD8+ T cell epitopes of VACV have been identified in mice, some of which are shared with ectromelia virus (ECTV). ECTV causes a systemic disease in mice, known as mousepox, that is an excellent small animal model for smallpox. Thus far work on the specificity of CD8+ T cell responses to VACV has shown that 1) the route of infection can affect the specificity of CD8+ T cell responses to VACV and 2) Not all epitopes are immunogenic in VACV strain MVA, an attenuated virus that is a candidate smallpox vaccine, despite their conservation in the MVA genome. In addition, evidence from other models of immunity to viruses suggest that not all CD8+ T cell epitopes are equally useful as vaccines. This application is based around these three observations. We will use well characterized mouse models for poxvirus vaccination and disease to establish general principles that will help us use analogous epitope information from human studies appropriately. In aim 1, different routes of VACV immunization will be tested to determine the extent to which route affects the specificity of CD8 T cell responses to VACV and if these route-related differences alter efficacy of protection against mousepox. Aim 2 tests a) the immunogenicity of conserved CD8+ T cell epitopes in a variety of VACV smallpox vaccines and b) whether these epitopes are presented on cells infected with VACV, ectromelia and monkeypox viruses. Aim 3 will use peptides representing individual epitopes to test if all are equally protective and also test the effect of removing or adding immunodominant epitopes to VACV vaccines or mousepox challenge viruses to see how important these dominant specificities are for vaccine efficacy. The possibility of smallpox being used as a bioweapon and the emergence of poxvirus diseases such as monkeypox show that poxviruses are a threat to human health. The current smallpox vaccine is unsafe and needs replacement. This study will provide basic information about the immune response to poxviruses and will help us know how best to use immunological knowledge and tools to design and test new smallpox: vaccines. [unreadable] [unreadable] [unreadable]